Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

Anti–tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis

Objective

Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)–blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti‐TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF‐κB ligand (RANKL) in synovial tissue.

Methods

The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double‐blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one‐way analysis of variance, with Tukey's post hoc test.

Results

Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF‐primed osteoblasts and endothelial cells.

Conclusion

Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti‐TNF therapy.

     

Modulation of RANKL and osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis in response to disease-modifying antirheumatic drug treatment and correlation with radiologic outcome

OBJECTIVE: To demonstrate the effect of treatment with disease-modifying agents on the expression of osteoprotegerin (OPG) and RANKL in the synovial tissue from rheumatoid arthritis (RA) patients and to correlate these changes with radiologic damage measured on sequential radiographs of the hands and feet. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 25 patients with active RA (16 of whom had a disease duration <12 months) before and at 3-6-month intervals after starting treatment with a disease-modifying agent. Immunohistologic analysis was performed using monoclonal antibodies to detect OPG and RANKL expression, with staining quantitated using computer-assisted image analysis and semiquantitative analysis techniques. Serial radiographs of the hands and feet were analyzed independently by 2 radiologists and a rheumatologist using the van der Heide modification of the Sharp scoring method. RESULTS: Thirteen patients achieved a low disease state as defined by a disease activity score <2.6 while 19 patients achieved an American College of Rheumatology response >20% after disease-modifying antirheumatic drug (DMARD) treatment. Successful DMARD treatment resulted in an increase in OPG expression and a decrease in RANKL expression at the synovial tissue level, which correlated with a reduction in erosion scores measured on annual radiographs of the hands and feet. CONCLUSION: Successful treatment-induced modulation of OPG and RANKL expression at the synovial tissue level, resulting in a reduction in the RANKL:OPG ratio, is likely to have a significant impact on osteoclast formation and joint damage in patients with active RA.     

OPG RANKL RANK等因子在类风湿关节炎患者血清及滑膜液中的变化及意义

目的检测类风湿关节炎（RA）患者血清、滑膜液中骨保护素（OPG）、核刺激因子受体配体（RANKL）、核刺激因子受体（RANK）、白细胞介素（IL）-18和前列腺素E-2（PGE2）水平，探讨其与RA发生发展的相关意义。方法采用酶联免疫吸附试验（ELISA）检测60例RA患者与60例同期入院骨外伤患者（作为正常对照组）血清、滑膜液中OPG、RANKL、RANK、IL-18和PGE2水平，分析其与RA的相关性。结果RA患者血清、滑膜液中OPG水平明显低于正常对照组（P〈0．05），RANKL、RANK、IL-18和PGE2水平明显高于对照组（p〈0．05）。结论RA患者血清、滑膜液中OPG水平降低，RANKL、RANK、IL-18和PGE2水平升高。推测上述细胞因子参与RA发病过程。     

Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis

OBJECTIVE: Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)-blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti-TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in synovial tissue. METHODS: The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double-blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one-way analysis of variance, with Tukey's post hoc test. RESULTS: Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF-primed osteoblasts and endothelial cells. CONCLUSION: Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti-TNF therapy.     

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade

The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-κβ ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 μg/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.     

Variability of RANKL and osteoprotegerin staining in synovial tissue from patients with active rheumatoid arthritis: quantification using color video image analysis

OBJECTIVE: To assess the interpatient, interbiopsy, and intrabiopsy variability of receptor activator of nuclear factor kB ligand (RANKL) and osteoprotegerin (OPG) immunostaining within synovial tissue from rheumatoid knee joints with active synovitis, using digital image analysis. METHODS: Synovial biopsy specimens were obtained from patients with rheumatoid arthritis (RA) and active synovitis. Immunohistologic analysis was performed on frozen synovial tissue biopsy specimens from 6 patients using a monoclonal antibody (Mab) to detect RANKL (626) or OPG (805 or 8051). Patients with a minimum of 4 synovial biopsies were included in the study. Sections were evaluated by computer assisted image analysis to assess between-patient, between-biopsy, and intra-biopsy variability of OPG and RANKL protein expression. The study was designed to deliberately maximize the variability. RESULTS: Computerized image analysis of staining with Mab to RANKL and OPG revealed variance for each antibody across the 3 components of the total variability. CONCLUSION: Our study shows that variability in synovial immunostaining of RANKL and OPG protein is a significant and complex problem. We discuss methods to reduce this variability and suggest that the auspices of OMERACT may be employed to advance the study of synovium in collaborative international studies.     

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.     

The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation

OBJECTIVE: Spondylarthritis (SpA) and rheumatoid arthritis (RA) have different patterns of bone damage, with more pronounced bone erosions in RA. The RANK/RANKL/osteoprotegerin (OPG) system plays a central role in bone resorption by promoting the maturation and activation of osteoclasts. To assess the potential role of this system in the distinct bone phenotype, we studied the synovial expression of these mediators in SpA and RA peripheral synovitis. METHODS: Synovial biopsy specimens were obtained from the actively inflamed peripheral joints of 35 patients with SpA and 19 patients with RA. Paired synovial biopsy samples were obtained from 24 patients with SpA after tumor necrosis factor alpha (TNFalpha) blockade. Synovial tissue sections were immunostained for RANKL, OPG, RANK, and TRAP and assessed by semiquantitative scoring and digital image analysis. RESULTS: After extensive validation of the reactivity and specificity of the antibodies, we demonstrated the abundant expression of RANKL and OPG in SpA synovitis. RANKL was expressed by both fibroblast-like synoviocytes and sublining T lymphocytes. RANK-positive osteoclast precursors but no mature TRAP-positive osteoclasts were present in the inflamed tissue. The expression of these mediators was not different between patients with nonpsoriatic SpA, patients with psoriatic SpA, and patients with RA, was not related to the degree of systemic or local inflammation, and was not significantly modulated by highly effective treatment with TNFalpha blockers. Only the subset of patients with the best systemic response to TNFalpha blockade had decreased RANKL expression in the intimal lining layer. CONCLUSION: The relative protection against bone erosions in SpA cannot be explained by qualitative or quantitative differences in the synovial expression of RANKL, OPG, and RANK. The abundant expression of these factors in SpA peripheral synovitis is largely disconnected from systemic and local inflammation.     

Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.     

Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis

We examined OPG and soluble RANKL in the serum (sOPG, sRANKL) and synovial fluid (synOPG, synRANKL) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OPG and RANKL were measured in 85 patients (44 with RA, 41 patients with OA) in serum and synovial fluid as well. For measuring of OPG and RANKL ELISA tests were used. The results of OPG and RANKL were compared with clinical and radiological scores. We found a negative correlation for OPG and RANKL in synovial fluids: not only for the whole group of patients (P< 0.003, r=–0.32), but also for the subgroups (RA: P<0.04, r=–0.28, OA: P<0.002, r=–0.54). SRANKL and synRANKL were positively correlated in the whole group (P<0.01, r=0.25) and in the OA group (P<0.02, r=0.35); the RA group was showing a trend (P<0.063, r=0.24), however. Serum OPG was lower in RA, synOPG higher in OA. The difference between the two patient groups was only significant for synOPG (P<0.03, r=0.056), but not for sOPG (P<0.09, r=0.19), sRANKL (P<0.43, r=0.85) or synRANKL (P<0.11, r=0.22). The synOPG:synRANKL ratio was significantly correlated with the Larsen score (P<0.004, r=0.38). Synovial OPG is significantly decreased in rheumatoid joints, whereby synovial RANKL is increased. Lower synOPG could reflect a lower protective effect on bone, thus leading to an earlier and more pronounced bone destruction in RA. However, the effect of different mediators for joint destruction in RA and OA seems not to be important to the pathophysiological changes in the joints. The upregulation of serum OPG might be the result of the inflammation; in contrast, an upregulation of RANKL could not be found in the serum of patients with RA and OA.     

Peripheral blood T lymphocytes from patients with early rheumatoid arthritis express RANKL and interleukin‐15 on the cell surface and promote osteoclastogenesis in autologous monocytes

Objective

To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process.

Methods

T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate‐resistant acid phosphatase staining and calcified matrix resorption activity.

Results

Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin‐15 (IL‐15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL‐15, IL‐17, tumor necrosis factor α (TNFα), and IL‐1β. OPG, anti‐TNFα, and anti–IL‐1β demonstrated a cooperative inhibitory effect. At 1‐year followup, surface RANKL and IL‐15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment.

Conclusion

T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.

     

Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients

OBJECTIVE: To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG). METHODS: Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. RESULTS: Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. CONCLUSION: The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.     

High levels of osteoprotegerin and soluble receptor activator of nuclear factor κB ligand in serum of rheumatoid arthritis patients and their normalization after anti–tumor necrosis factor α treatment

Objective

To test the hypotheses that 1) proinflammatory cytokines affect osteoprotegerin (OPG) and soluble receptor activator of nuclear factor κB ligand (sRANKL) production and therefore the OPG and sRANKL levels differ in rheumatoid arthritis (RA) patients in comparison with healthy individuals; and 2) anti–tumor necrosis factor α (anti–TNFα) therapy influences OPG and sRANKL levels.

Methods

Sera were obtained from healthy individuals or RA patients receiving the combination of infliximab and methotrexate. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated from RA patients. Fibroblast‐like synoviocytes (FLS) were isolated from synovial tissue obtained at total knee replacement in RA patients. Supernatants from cells stimulated with cytokines were collected after culture in vitro. Concentrations of OPG and sRANKL were determined by enzyme‐linked immunosorbent assays.

Results

A strong positive correlation between OPG concentration and age was observed in healthy individuals but not in RA patients. The OPG and sRANKL levels were higher in RA patients than in healthy controls. Cultured FLS spontaneously secreted much higher amounts of OPG than PBMCs or SFMCs. Proinflammatory cytokines enhanced OPG production. Anti‐TNFα treatment resulted in the normalization of serum OPG and sRANKL levels in RA patients without influencing the OPG:sRANKL ratio.

Conclusion

Although higher serum levels of OPG and sRANKL are present in RA patients than in healthy individuals, the ratio of OPG:sRANKL is similar. There is an age‐dependent increase of OPG but not sRANKL levels in healthy subjects. Anti‐TNFα treatment results in the normalization of elevated levels of OPG and sRANKL in RA patients.

     

Regenerating gene (REG) 1 alpha promotes pannus progression in patients with rheumatoid arthritis

Introduction

A protein analysis using mass spectrometry revealed the existence of serum proteins with significant quantitative changes after the administration of infliximab. Among these proteins, regenerating gene (REG) 1?? appears to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, the present study was conducted to examine the mechanism of REG1?? in RA disease progression.

Methods

Serum samples were collected from RA patients and normal healthy controls. REG1?? expression was evaluated by ELISA, RT-PCR, and indirect immunofluorescence microscopy. The functions of REG1?? on synovial fibroblasts with regard to apoptosis, receptor activator of NF-??B ligand (RANKL) expression, and cellar proliferation were evaluated using siRNA to inhibit the intrinsic REG1?? mRNA expression.

Results

The serum concentrations of REG1?? in RA patients were higher than in normal healthy controls. The high expression of REG1?? was also observed in the synovial tissue of RA patients compared to those of osteoarthropathy patients. In addition, tumor necrosis factor-?? (TNF-??) upregulated REG1?? expression in the synovial fibroblasts cell line (MH7A). Inhibition of REG1?? expression suppressed the induction of RANKL expression by TNF-??. Furthermore, exogenous recombinant REG1?? protein inhibited apoptosis and promoted cell proliferation in MH7A cells. These effects were abolished in the REG1??-siRNA MH7A cells.

Conclusion

The present data suggest that TNF-?? induces aberrant REG1?? expression and that REG1?? plays an important role in aberrant cell proliferation and RANKL expression of synovial fibroblasts, ultimately resulting in pannus formation. Restoration of normal physiological REG1?? expression may contribute to disease amelioration.     

Baseline serum RANKL levels may serve to predict remission in rheumatoid arthritis patients treated with TNF antagonists

Aims

The objective of this study was to investigate whether baseline receptor activator for nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) serum (s) levels can predict the therapeutic response to TNF antagonists (a‐TNF).

Methods

We studied 75 rheumatoid arthritis patients (81% female) with a longstanding refractory disease. The variables of disease activity, physical function and sRANKL and sOPG levels were determined before and after both 12–14 and 28–30 weeks of a‐TNF therapy (65 adalimumab, 10 infliximab). Remission was defined by a 28 joint count disease activity score (DAS28) ⩽2.6 and clinical response by a reduction in DAS28⩾1.2 at both 3‐ and 7‐month follow‐up visits.

Results

In most patients, disease activity was severe, as reflected by a baseline DAS28 score of 5.9±1 (mean±SD), an HAQ of 1.6 (1.1 to 2.1) (median (interquartile range (IQR))) and a CRP 15 mg/l (IQR: 9 to 24). The sRANKL levels and RANKL/OPG ratio in patients that achieved remission were significantly lower at baseline than in the remaining patients at both 3 and 7 months of follow‐up. The sOPG levels correlated with the HAQ and the physician''s disease assessment and diminished significantly after a‐TNF treatment. However, no significant association was detected between the therapeutic response profile and sOPG levels.

Conclusions

These data suggest that in patients receiving a‐TNF treatment, lower serum levels of RANKL and RANKL/OPG ratio may serve to predict remission.In rheumatoid arthritis (RA), bone destruction in the erosions is mainly due to the abnormal activation of osteoclasts. It is currently known that the control of osteoclast formation and activation is principally mediated by three components of the TNF signalling pathway: the receptor activator for nuclear factor kappaB (RANK), a membrane‐bound osteclast receptor that initiates osteclastic bone resorption after binding its ligand (RANKL) and osteoprotegerin (OPG), the key inhibitor of bone resorption that acts as a soluble, non‐signalling decoy receptor for RANKL.¹ Therefore, the balance between OPG and RANKL is a fundamental factor in controlling bone resorption and can be influenced by several hormones and cytokines, including TNF.Soluble OPG and RANKL can be measured in human serum, and higher levels of these mediators have been described in serum samples from RA patients when compared with healthy individuals.² Moreover, higher levels of RANKL are also expressed in tissues from patients with active RA and spondyloarthropathies than in tissues from patients with inactive RA or osteoarthritis and from normal subjects.³ Given the success of TNF blockade in preventing structural damage in RA, some recent studies have focused on the effect of a‐TNF therapies on the soluble or tissue levels of RANKL and OPG.^2,4,5 However, to date, there are still no data to indicate the value of the levels of these proteins in predicting the therapeutic response. The aim of the present study was to investigate the utility of serum (s)RANKL and sOPG to predict a therapeutic response to a‐TNF.     

Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathy, osteoarthritis,and from normal patients: semiquantitative and quantitative analysis

OBJECTIVES: To compare receptor activator of NF-kappaB ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues. METHODS: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL. RESULTS: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA). CONCLUSIONS: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.